This competitive renewal proposes to continue our investigation of allosteric enhancers (AEs) of the A1 adenosine receptor (A1AR). These compounds bind to an allosteric site on the receptor to increase the affinity of adenosine to the orthosteric site on the A1AR. We have identified new and improved aminothiophene AEs and we have discovered a new chemical class of more potent and effective aminothiazole AEs. We also have discovered that some disulfides and H2O2 have AE-like effects on the A1AR and the A2AAR. To learn more about the molecular mechanisms of AE action we investigated receptor mutants and chimera. We have developed a novel system to "score" enhancer activity based on the ability of these compounds to prevent rapid dissociation of an agonist radioligand, 125I-ABA, from the A1AR in response to GTPgammaS. We propose new studies to achieve three specific aims. Aim 1 is to synthesize new enhancer structures in two chemical classes and to characterize them based on radioligand binding to receptors and guanine nucleotide exchange from G proteins. This aim is guided by conformational molecular field analysis (CoMFA) of existing structures and the use of new synthetic techniques. Aim 2 is to efficiently evaluate enhancer function in intact cells and to evaluate selected compounds for activity in modulating adenosine A1 receptor function in brain slices and stimulation of angiogenesis. Aim 3 will investigate the role and disulfide bond formation in AE and H2O2 action through analysis of thiol alkylating agents, epitope-tagged receptors, site-directed mutagenesis and protein sequencing. We will also investigate the possibility the H2O2 is a physiological regulator of A2AAR signaling. We hypothesize that cysteines in the extracellular loops of the A1AR participate in disulfide cross linking reactions. AEs are significant therapeutic candidates that may reduce chronic pain or promote site-specific angiogenesis. Moreover, our investigation of the role of thiols/disulfides in enhancer and H2O2 action may reveal important new information about general mechanisms of GPCR G protein coupling and trafficking.